Inhibitory effect of 10-hydroxy- trans -2-decenoic acid on LPS-induced IL-6 production via reducing IκB-ζ expression

Abstract

The effect of 10-hydroxy-trans-2-decenoic acid (10H2DA), a major fatty acid component of royal jelly, was investigated on LPS-induced cytokine production in murine macrophage cell line, RAW264 cells. 10H2DA inhibited LPS-induced IL-6 production dose-dependently, but did not inhibit TNF-α production. 10H2DA inhibited LPS-induced NF-κB activation in a dose-dependent fashion. In addition, NF-κB activation induced by over-expression of either MyD88 or Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) was also inhibited by 10H2DA. Degradation of IκB-α and phosphorylation of IκB kinase-α were not inhibited by 10H2DA. On the other hand, reduction of LPS-induced IκB-ζ expression was discovered. Production of lipocalin-2 and granulocyte colony-stimulating factor (G-CSF), which is dependent on IκB-ζ, was also inhibited by 10H2DA, whereas that of IκB-ζ–independent cytokines/chemokines, such as IFN-β, murine monocyte chemotactic protein-1 (JE), macrophage inflammatory protein (MIP)-1α and MIP-2, was not. Together, 10H2DA specifically inhibited LPS-induced IκB-ζ expression, followed by inhibition of IκB-ζ-dependent gene production. These results suggest that 10H2DA is one of the components of royal jelly to show anti-inflammatory effects and could be a therapeutic drug candidate for inflammatory and autoimmune diseases associated with IκB-ζ and IL-6 production.

Keywords

Fatty acid IκB-ζ IL-6 royal jelly Toll-like receptor

Introduction

TLRs are innate immune receptors recognizing microbial components such as bacterial LPS.¹ The TLR4/MD-2 complex recognizes LPS and its signaling activates NF-κB in two independent pathways, such as MyD88-dependent and Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF)-dependent pathways.²

As many inflammatory cytokines and chemokines are produced under the regulation of NF-κB, activation of NF-κB is strictly regulated in several ways.^3,4 Nuclear factor-κB proteins are homodimers or heterodimers of the NF-κB family including p65 and p50. IκB-ζ, a member of IκB family, can be a component of an active NF-κB complex, especially in activating IL-6 promoter induced by LPS.⁵ NF-κB p65 is sequestered in the cytoplasm through association with IκB in unstimulated cells. During LPS stimulation, p65 is released from IκB-α, a typical member of IκB, followed by nuclear translocation of the p50/p65 heterodimer.

Royal jelly is produced by the hypopharyngeal and mandibular glands of worker honeybees as a food essential for the growth of the queen honeybee. It has various pharmacological activities, such as vasodilative hypotensive activities, decreasing activity of serum cholesterol levels, anti-tumor activities, protective activity against hemopoietic dysfunction in X-irradiated mice, anti-allergic activities of reducing IgE production and anti-inflammatory activities in inhibiting pro-inflammatory cytokine production.⁶^–¹⁰ Royal jelly contains 10-hydroxy-trans-2-decenoic acid (10H2DA) as major lipid component,¹¹ which is a natural ingredient almost uniquely found in royal jelly. Therefore, it is possible that 10H2DA is an active component of royal jelly’s pharmacological activities. Indeed, anti-tumor and antibiotic activities have been reported for 10H2DA.^8,12 However, the molecular mechanisms for the pharmacological activities of 10H2DA are not well known.

In this study, we investigated the effect of 10H2DA on LPS-induced cytokine production.

Materials and methods

Reagents

10H2DA with a purity of above 98% (analyzed by high performance liquid chromatography), was kindly gifted by Api Co. (Gifu, Japan). It was dissolved in ethanol at 500 mM and added to culture media at the indicated concentration. LPS from Escherichia coli O55 was obtained from Sigma-Aldrich (St Louis, MO, USA). Antibodies against IκB-α (Sigma-Aldrich), IκB kinase (IKK)-α (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and phospho-Ser180 IKK-α (Cell Signaling Technology, Danvers, MA, USA) were used.

Plasmids

Murine Toll/IL-1 receptor domain-containing adaptor protein (TIRAP) gene was amplified from mRNA of RAW264 by RT-PCR and cloned into pENTR/TEV/D-TOPO (Invitrogen, Carlsbad, CA, USA). It was then transferred into pDEST26 (Invitrogen), as recommended by the manufacturer. The MyD88 expression vector¹³ was kindly gifted by Dr Takaoka (University of Tokyo), whereas TRIF¹⁴ was from Dr Komatsu (Aichi Medical University). Promoter regions of TNF-α, IL-6, and IκB-ζ gene were amplified from the genomic DNA of RAW264 by PCR and cloned into a luciferase reporter plasmid, pGL4.26 (Promega, Madison, WI, USA). An NF-κB reporter gene plasmid, pNF-κB-TA-luc, was purchased from Clontech (Palo Alto, CA, USA). A transfection control reporter plasmid, pRL-TK, was obtained from Promega. Plasmids were extracted with GenElute HP endotoxin-free plasmid Midiprep kit (Sigma-Aldrich) for transfection into cultured cell lines.

Cell culture, 10H2DA treatment and stimulation

The RAW264 murine macrophage cell line was obtained from RIKEN Bioresource Center (Tokyo, Japan) and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium containing 5% heat-inactivated fetal bovine serum (FBS; Invitrogen). The cells were seeded in a 96-well plate at 2 × 10⁵ cells/well for cytokine assays or 2.5 × 10⁴ cells/well for reporter gene assays. The cellswere pre-incubated with or without the indicatedconcentration of 10H2DA for 30 min then stimulated by adding LPS at the final concentration of 100 ng/ml. After 24 h incubation, the culture supernatants wereused for cytokine assays. The human embryonic kidney (HEK) cell line HEK293 was maintained in minimal essential medium containing 5% FBS and 1% nonessential amino acid (Sigma-Aldrich). The HEK293 cells were seeded in a 96-well plate at a cell density of2.5 × 10⁴ cells/well for reporter gene assays. At theend of each assay, cell viability was assayed by AlamarBlue (Trek Diagnostic Systems, West Sussex, UK), as recommended by the manufacturer. In all experimental conditions performed in this study, cell viability was >95% compared with untreated cells.

Analysis for cytokines/chemokines expression by RT-PCR

RAW264 cells were cultured on six-well plate and stimulated with LPS. The cells were harvested and total RNA was extracted by RNeasy mini kit (QIAGEN, Valencia, CA, USA), as recommended by the manufacturer. The RNA was reverse-transcribed with ReverTraPlus (Toyobo, Tokyo, Japan). Each cytokine/chemokine cDNA was amplified with specific primers as listed in Table 1¹⁵^–¹⁹ for each mRNA by SYBR Green Real-time PCR Master mix (Toyobo). Amplified PCR products were analyzed with both iCycler iQ real-time PCR detection system (Bio-Rad Laboratories, Tokyo, Japan) and on agarose gel electrophoresis followed by ethidium bromide staining.

Table 1.

Primers used for RT-PCR analysis

Gene(product; bp)		Primer sequence	Reference
IL-6	(270)	TCC ATC CAG TTG CCT TCT TG	This study
IL-6	(270)	ATC CAT CAT TCT TTG TAT C	This study
TNF-α	(374)	TTG ACC TCA GCG CTG TTG	15
TNF-α	(374)	CCT GTA GCC CAC GTC GTA GC	15
IFN-β	(312)	AAA CAA TTT CTC CAG CAC TG	16
IFN-β	(312)	ATT CTG AGG CAT CAA CTG AC	16
IκB-ζ	(807)	ACA TCA CCG CAA ACG CCT ACA CCT G	17
IκB-ζ	(807)	CGT GTC ACC ATC TCC GTC CCT AGC G	17
Lipocalin-2	(100)	TGC CAC TCC ATC TTT CCT GTT	This study
Lipocalin-2	(100)	GGG AGT GCT GGC CAA ATA AG	This study
G-CSF	(206)	CAC CCA GTT CTG TGG CAG TA	This study
G-CSF	(206)	TCC TGT GAG GAC AGG AAA CC	This study
JE	(374)	CCC AGC CAG ATG CAG TTA ACG CCC CAC T	18
JE	(374)	TTC ACT GTC ACA CTG GTC ACT C	18
MIP-1α	(258)	CAC CCT CTG TCA CCT GCT CAA CAT C	18
MIP-1α	(258)	GGT TCC TCG CTG CCT CCA AGA CTC T	18
MIP-2	(285)	CCG CTG TTG TGG CCA GTG AAC TGC G	18
MIP-2	(285)	TTA GCC TTG CCT TTG TTC AGT AT	18
HPRT	(177)	GTA ATG ATC GTC AAC GGG GGA C	19
HPRT	(177)	CCA GCA AGC TTG CAA CCT TAA C	19

G-CSF, granulocyte-colony stimulating factor; JE, murine monocyte chemotactic protein-1; MIP, macrophage inflammatory protein; HPRT, hypoxanthine phosphoribosyltransferase.

Cytokine analysis

The TNF-α and IL-6 concentrations in the culture supernatant were analyzed by conventional sandwich ELISA. Antibodies and recombinant cytokines for ELISA were purchased from e-Bioscience (San Diego, CA, USA).

Reporter gene assay

Cells seeded in wells of a 96-well plate were transfected with a luciferase reporter construct and pRL-TK plasmid using FuGENE-HD (Roche, Indianapolis, IN, USA) for RAW264 or Lipofectamine 2000 (Invitrogen) for HEK293, as recommended by the manufacturer. After indicated treatment, the cells were lysed with Passive Lysis Buffer (Promega). Luciferase activity in the cell lysate was determined by Dual-Luciferase Reporter assay system (Promega).

Immunoblotting

The RAW264 cells were pretreated with 10H2DA (4 mM) for 30 min then cultured with LPS (100 ng/ml). The cells were harvested and lysed in 50 mM Tris-HCl buffer (pH 7.5) containing 0.5% Nonidet P-40, 150 mM NaCl, 5 mM EDTA containing 1 mM phenylmethylsulfonyl fluoride (PMSF), and phosphatase inhibitor cocktail II (Sigma-Aldrich). The lysate was centrifuged at 20,000 g for 15 min and the supernatant used as cytoplasmic protein. After boiling at 100°C for 5 min, equal amounts (20 µg/lane) of cytoplasmic protein were loaded onto a 8% gel, run under reducing conditions and transferred onto Immobilon Transfer Membrane (Millipore, Bedford, MA, USA). The membranes were treated with 5% skimmed milk for 1 h, rinsed with 25 mM Tris-HCl buffer (pH 7.4) containing 137 mM NaCl and 2.7 mM KCl, and incubated with a 1:1000 dilution of an antibody for 1 h. The blots were further treated with a 1:5000 dilution of HRP-conjugated anti-rabbit IgG (Thermo Scientific, Rockford, IL, USA) for 1 h. The immune complexes on the blots were detected by SuperSignal West Dura system (Pierce, Rockford, IL, USA) and LAS-3000UVmini (Fujifilm, Tokyo, Japan).

Statistical analysis

Results are expressed as means ± SD. Statistical analyses were performed using GraphPad PRISM software (GraphPad, San Diego, CA, USA). Significant differences between control and 10H2DA treatment were evaluated by Dunnett’s multiple comparison test after one-way ANOVA and by Student’s t-test. A P-value of <0.05 was considered statistically significant.

Results

10H2DA inhibited LPS-induced IL-6 but not TNF-α production

10H2DA significantly inhibited IL-6 but not TNF-α production at concentrations 2–4 mM (Figure 1A). Both TNF-α and IL-6 were produced upon LPS stimulation but in different time-courses (Figure 1B). By adding 4 mM of 10H2DA, IL-6 production was significantly inhibited, whereas TNF-α production was not. Total RNA extracted from cells stimulated with LPS in the presence, or absence of, 4 mM 10H2DA over 24 h and analyzed by semi-quantitative and quantitative RT-PCR also revealed that IL-6, but not TNF-α, mRNA expression was obviously decreased (Figure 2).

Figure 1.

Effect of 10H2DA on LPS-induced IL-6 and TNF-α production. RAW264 cells were incubated with 0, 1, 2, or 4 mM (a) or 4 mM (b) 10H2DA for 30 min followed by stimulation with 100 ng/ml of LPS for 24 h (A) or the indicated periods (B). Concentrations of IL-6 and TNF-α in culture supernatants were measured by ELISA. Data are expressed as mean ± SD (N = 3). ND, not detected. ^*P < 0.001 compared with no 10H2DA. Open column and circle, without 10H2DA; filled column and circle, with 10H2DA.

Figure 2.

Effect of 10H2DA on LPS-induced IL-6 and TNF-α mRNA expression. RAW264 cells were incubated with or without 4 mM 10H2DA for 30 min followed by stimulation with 100 ng/ml of LPS for the indicated periods. Total RNA extracted from the cells was subjected to semi-quantitative RT-PCR (A) or quantitative RT-PCR (B) using specific primers. The HPRT mRNA levels were used as control (A) and for normalization of data (B). Data are representative of ≥2 independent experiments with similar results. Δ, 10H2DA alone; ○, LPS alone; •, LPS + 10H2DA.

10H2DA inhibited NF-κB activation

In RAW264 cells NF-κB activation with LPS was dose-dependently inhibited by 1–4 mM of 10H2DA (Figure 3A). NF-κB activation induced by over-expression of MyD88, TIRAP and TRIF was also significantly inhibited by 4 mM 10H2DA (Figure 3B).

Figure 3.

Effect of 10H2DA on NF-κB activation in LPS signaling. (A) RAW264 cells transfected with pNF-κB-TA-luc (100 ng/well) and pRL-TK (10 ng/well) were incubated with (filled column) or without (open column) the indicated concentration of 10H2DA for 30 min followed by stimulation with 100 ng/ml LPS for 6 h. Luciferase activities in cell lysates were measured, normalized and expressed as fold induction compared with unstimulated control. Data are expressed as mean ± SD (N = 3). *P < 0.05 compared with no 10H2DA. (B) HEK293 cells were transfected with 10 ng/well of control vector pEF (mock) or expression plasmid for MyD88, murine TIRAP, or TRIF. At the same time, pNF-κB-TA-luc (10 ng/well) and pRL-TK (1 ng/well) were co-transfected. Five h after transfection, culture media were exchanged for media containing the indicated concentration of 10H2DA (filled column) or PBS (open column) and the cells were further incubated for 24 h. Luciferase activities in cell lysates were determined. NF-κB activation was expressed as fold induction of luciferase activity compared with that of mock transfectant. Data are expressed as mean ± SD (N = 3). **P < 0.01; ***P < 0.001 compared with no 10H2DA.

Immunoblotting revealed that 10H2DA did not inhibit LPS-induced IKK-α phosphorylation and IκB-α degradation (Figure 4).

Figure 4.

Effect of 10H2DA on LPS-induced IKK-α phosphorylation and IκB-α degradation. RAW264 cells were incubated with 4 mM 10H2DA for 30 min followed by stimulation with 100 ng/ml of LPS for the indicated periods. An equal amount of cytoplasmic protein (20 µg) obtained from the cells was separated by SDS-PAGE and visualized by immunoblotting analysis. Data are representative of ≥2 independent experiments with similar results.

10H2DA inhibited (IκB)-ζ mRNA expression

Analyses by RT-PCR revealed that, under LPS stimulation, expression of IκB-ζ mRNA was increased at 3 h (Figure 5). 10H2DA, on the other hand, inhibited expression of IκB-ζ mRNA.

Figure 5.

Effect of 10H2DA on LPS-induced IκB-ζ mRNA transcription. RAW264 cells were incubated with or without 4 mM 10H2DA for 30 min followed by stimulation with 100 ng/ml of LPS for the indicated periods. Total RNAs extracted from the cells were subjected to semi-quantitative (A) and quantitative (B) RT-PCR using specific primers. The HPRT mRNA levels were used as control (A) and for normalization of data (B). Data are representative of ≥2 independent experiments with similar results. Δ, 10H2DA alone; ○, LPS alone; •, LPS + 10H2DA.

10H2DA inhibited IL-6 and IκB-ζ but not TNF-α transcription induced by LPS and TLR signaling molecules

According to luciferase reporter gene assay, in RAW264 cells LPS induced activation of TNF-α, IL-6 and IκB-ζ promoters (Figure 6). As expected, LPS-induced promoter activity of IL-6 and IκB-ζ, but not TNF-α, was dose-dependently inhibited by 10H2DA (Figure 6A). Furthermore, IκB-ζ promoter activation induced by over-expression of MyD88 was also significantly inhibited by 10H2DA (Figure 6B).

Figure 6.

Effect of 10H2DA on LPS-induced TNF-α, IL-6, and IκB-ζ promoter activation. (A) Luciferase reporter construct of TNF-α, IL-6, or IκB-ζ promoter and pRL-TK was transfected into RAW264 cells. The cells were incubated with (filled column) or without (open column) the indicated concentration of 10H2DA for 30 min followed by stimulation with 100 ng/ml of LPS for 6 h (TNF-α andIκB-ζ) or 12 h (IL-6). Luciferase activity in cell lysates was measured, normalized and expressed as fold induction, compared withunstimulated control. Data are expressed as mean ± SD (N = 3). *P < 0.05; **P < 0.01 compared with no 10H2DA. (B) Luciferase reporter construct of IκB-ζ promoter was co-transfected with MyD88-expressing plasmid into HEK293 cells. Five h after transfection,10H2DA was added at the indicated concentration. After 6 h incubation, luciferase activity in cell lysates were measured,normalized and expressed as fold induction, compared with mock-transfectant without 10H2DA. Data are expressed as mean ± SD (N = 3). *P < 0.05; **P < 0.01; ***P < 0.001 compared with no 10H2DA. Open column, without 10H2DA; filled column, with 10H2DA.

10H2DA inhibited IκB-ζ–dependent gene expression

Expression of lipocalin-2 and G-CSF was dependent on IκB-ζ, in comparison with independent genes IFN-β, murine monocyte chemotactic protein-1 (JE), MIP-1 and MIP-2. The LPS-induced expression of IκB-ζ–dependent genes lipocalin-2 and G-CSF was significantly inhibited by 10H2DA, whereas that of IFN-β, JE, MIP-1 and MIP-2 was not (Figure 7).

Figure 7.

Effect of 10H2DA on cytokines/chemokines mRNA expression. The RAW264 cells were incubated with or without 4 mM 10H2DA for 30 min followed by stimulation with 100 ng/ml LPS for the indicated periods. Total RNA extracted from the cells was subjected to semi-quantitative RT-PCR (A) or quantitative RT-PCR (B) using specific primers. The HPRT mRNA levels were used as control (A) and for normalization of data (B). Data are representative of ≥2 independent experiments with similar results. Lipocalin-2 and G-CSF are IκB-ζ–dependent genes. Δ, 10H2DA alone; ○, LPS alone; •, LPS + 10H2DA.

Discussion

De novo synthesis of IκB-ζ has been reported to be critical for IL-6 production in LPS stimulation.⁵ After stimulation of IκB-ζ production with LPS through the MyD88-dependent pathway, IκB-ζ interacts with the p50 subunit of NF-κB, following which the active NF-κB complex upregulates IL-6 production.

We revealed specific inhibitory effects of 10H2DA on LPS-induced IκB-ζ production followed by inhibition of IκB-ζ–dependent gene expression, including IL-6 (Figure 8). IL-6 has been reported to play critical roles in colonic inflammation and colon cancer.²⁰ Recently, IL-6 was found to be a key contributor in the development of Th17 helper T cells, which critically contribute to autoimmune and inflammatory diseases.^21,22 Lipocalin-2 and G-CSF genes have also been reported to be IκB-ζ–dependent genes, as well as IL-6.²³ We detected significant inhibition of expression of these genes by 10H2DA, whereas no significant reduction was seen in the expression of IκB-ζ-independent genes, such as TNF-α, IFN-β, JE, MIP-1α and MIP-2, suggesting that a reduction in IκB-ζ expression results in the inhibition of IL-6 production. Lipocalin-2 has been suggested to promote insulin resistance.²⁴ Furthermore, IκB-ζ is involved in IFN-γ production by TNF-α stimulation.²⁵ Recently, IκB-ζ has been reported to positively regulate Th17 development in co-operation with ROR nuclear receptors.²⁶ IκB-ζ-deficient mice showed resistance to experimental autoimmune encephalomyelitis. IκB-ζ expression is supposed to contribute to these various pathological conditions. Therefore, 10H2DA may improve these pathological consequences via its activity in reducing IκB-ζ-induction and IκB-ζ–dependent gene-expression.

Figure 8.

A schematic of the effect of 10H2DA on LPS-stimulated gene expression. LPS induces expression of NF-κB-dependent genes, such as TNF-α and JE, through MyD88/NF-κB signaling pathway. IκB-ζ is one of the NF-κB-dependent genes and induces the IκB-ζ-dependent genes including IL-6, lipocalin-2 and G-CSF. 10H2DA acid inhibits only a subset of NF-κB, which activates IκB-ζ promoter and is suspected to be in a different state of phosphorylation or other post-translational modifications.

TNF-α and IL-6 are known to be major pro-inflammatory cytokines induced by MyD88-dependent LPS signaling.²⁷ NF-κB is a well known transcription factor responsible for IL-6 production.²⁸ In LPS stimulation, MyD88-dependent NF-κB activation is thought to be responsible for IκB-ζ gene expression.⁵ Using a reporter gene assay, 10H2DA inhibited NF-κB activation under LPS and MyD88 induction. In RT-PCR analysis, however, 10H2DA did not affect the production of cytokines or chemokines, including TNF-α and JE, whose expression is supposed to be dependent on NF-κB. Furthermore, promoter activation of IκB-ζ, but not the TNF-α gene, was inhibited by 10H2DA in reporter gene assay. The NF-κB reporter construct that we used has four repeats of the consensus κB sequence, which is identical to that in the promoter region of the IκB-ζ gene. The κB sequence-specific binding of a subset of NF-κB complexes may be the reason that expression of IκB-ζ, but not the other NF-κB-dependent genes, was inhibited by 10H2DA. In fact, we could not detect the inhibitory effect of 10H2DA on the degradation of IκB-α. LPS-stimulated IKK-α phosphorylation was even increased by adding 10H2DA. We could also not detect the inhibition of NF-κB p65 nuclear translocation by 10H2DA (data not shown). These data suggest that at least part of NF-κB was released from the IκBs and translocated into the nucleus in LPS stimulation. Therefore, some NF-κB-dependent genes could be transcribed.

NF-κB activation is controlled by IKKs which are required for phosphorylation-induced degradation of IκBs.³ Following ligation of TLRs, IKKs are activated by phosphorylation, resulting in IκB phosphorylation and degradation.³ We could not detect significant inhibition of IκB degradation by 10H2DA, suggesting that NF-κB was at least released from the IκB–NF-κB complex. Full activation of NF-κB needs several post-translational modifications, including phosphorylation.⁴ Activation of p65, a subunit of NF-κB activated in LPS stimulation, is believed to be regulated by phosphorylation of at least four Ser residues. Phosphorylation at Ser 276 is critical for interaction with the transcriptional co-activators cAMP response element-binding protein (CREB)-binding protein and p300,²⁹ and is therefore necessary for NF-κB–dependent gene transcription. The other phosphorylation sites may affect specific gene expression. Anrather et al.³⁰ reported that phosphorylation of Ser residues at 205, 276 and 281 is necessary for the expression of MIP-2 and ICAM-1, whereas IL-6 and vascular cell adhesion molecule (VCAM)-1 are expressed in a phosphorylation-independent manner. On the otherhand, Ser 536 needs to be phosphorylated to produce IL-6 in angiotensin II-stimulated vascular smooth muscle cells.³¹ They also reported significant amounts of NF-κB were present in the nucleus; however, unphosphorylated NF-κB does not activate gene transcription, even in the nucleus. Although we could not detect an inhibitory effect of 10H2DA on LPS-induced phosphorylation of p65 at Ser 536 (data not shown), regulation of p65 phosphorylation at specific Ser residues may allow modulation of particular gene expression, including the IκB-ζ gene.

Kohno et al.¹⁰ showed that royal jelly inhibited LPS- and IFN-γ–induced cytokine production, and identified one of the major protein components, MRJP3, as an anti-inflammatory factor derived from honeybee royal jelly.¹⁰ They also suggested that royal jelly contains another anti-inflammatory factor with low molecular mass that inhibits TNF-α and IL-6 production. Although we could not detect an inhibitory effect on LPS-induced TNF-α production, 10H2DA may be one of the low molecular weight anti-inflammatory factors in royal jelly.

We showed that 2–4 mM of 10H2DA inhibited LPS-induced IL-6 production from murine macrophage cells. Fresh and pure honeybee royal jelly has been reported to contain >100 mM of 10H2DA,¹¹ suggesting that >2 mM concentrations of 10H2DA is conceivable in gut mucosa when raw royal jelly is eaten. Furthermore, we could detect a modest inhibitory effect of 10H2DA, even by addition 6 h after LPS stimulation (data not shown), suggesting that continuous ingestion of 10H2DA in royal jelly may reduce chronic IκB-ζ-dependent gene expression. Together, 10H2DA in royal jelly could reach a high enough concentration, at least in gut lumen, to inhibit the activation of macrophages and other immune cells, and could be effective for expressing anti-inflammatory effects, especially for ongoing chronic inflammation in the intestine.

In conclusion, we demonstrated that 10H2DA inhibited LPS-induce IκB-ζ production followed by gene expression of IκB-ζ–dependent genes, including IL-6. This is, as far as we know, the first report of a specific inhibitor for IκB-ζ induction. Accumulating evidence that IκB-ζ and IL-6 contribute to inflammatory and autoimmune diseases suggests that 10H2DA may be a therapeutic drug candidate for IκB-ζ– and IL-6–dependent disorders.

Conflicts of interest

None declared.

Footnotes

Acknowledgements

We thank Dr Takaoka and Dr Komatsu for providing the plasmids. This work was supported by a grant from the Japan Royal Jelly Fair Trade Council (Tokyo, Japan) and partly by Grant-in-Aid for Challenging Exploratory Research from the Japan Society for the Promotion of Science [21659110 to TS].

References

Akira

Uematsu

Takeuchi

. Pathogen recognition and innate immunity. Cell 2006; 124: 783–801.

Yamamoto

Takeda

Akira

. TIR domain-containing adaptors define the specificity of TLR signaling. Mol Immunol 2004; 40: 861–868.

Hayden

Ghosh

. Shared principles in NF-κB signaling. Cell 2008; 132: 344–362.

Perkins

. Post-translational modifications regulating the activity and function of the nuclear factor κB pathway. Oncogene 2006; 25: 6717–6730.

Yamamoto

Yamazaki

Uematsu

. Regulation of Toll/IL-1-receptor-mediated gene expression by the inducible nuclear protein IκBζ. Nature 2004; 430: 218–222.

Fujii

Kobayashi

Kuboyama

. Augmentation of wound healing by royal jelly (RJ) in streptozotocin-diabetic rats. Jpn J Pharmacol 1990; 53: 331–337.

Helleu

. [Antibacterial properties of royal jelly; bactericidal and antibiotic effects of neutralized royal jelly.]. Ann Inst Pasteur (Paris) 1956; 91: 231–237.

Townsend

Morgan

Hazlett

. Activity of 10-hydroxydecenoic acid from royal jelly against experimental leukaemia and ascitic tumours. Nature 1959; 183: 1270–1271.

Okamoto

Taniguchi

Kunikata

. Major royal jelly protein 3 modulates immune responses in vitro and in vivo. Life Sci 2003; 73: 2029–2045.

10.

Kohno

Okamoto

Sano

. Royal jelly inhibits the production of proinflammatory cytokines by activated macrophages. Biosci Biotechnol Biochem 2004; 68: 138–145.

11.

G.Lercker

Capella

. S. Conte and Giordani FRaG. Components of royal jelly II. The lipid fraction, hydrocarbons and sterols. J Apic Res 1982; 21: 178–184.

12.

Blum

Novak

Taber

S III

. 10-Hydroxy-Δ 2-decenoic acid, an antibiotic found in royal jelly. Science 1959; 130: 452–453.

13.

Honda

Yanai

Mizutani

. Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling. Proc Natl Acad Sci USA 2004; 101: 15416–15421.

14.

Komatsu

Takeuchi

Gotoh

. Bovine parainfluenza virus type 3 accessory proteins that suppress β interferon production. Microbes Infect 2007; 9: 954–962.

15.

Tucker

Sack

Jr . Expression of serum amyloid A genes in mouse brain: unprecedented response to inflammatory mediators. FASEB J 2001; 15: 2241–2246.

16.

Sugiyama

Fujita

Koide

. 2-aminopurine inhibits lipopolysaccharide-induced nitric oxide production by preventing IFN-β production. Microbiol Immunol 2004; 48: 957–963.

17.

Motoyama

Yamazaki

Eto-Kimura

Takeshige

Muta

. Positive and negative regulation of nuclear factor-κB-mediated transcription by IκB-ζ, an inducible nuclear protein. J Biol Chem 2005; 280: 7444–7451.

18.

Tessier

Naccache

Clark-Lewis

Gladue

Neote

McColl

. Chemokine networks in vivo: involvement of C-X-C and C-C chemokines in neutrophil extravasation in vivo in response to TNF-α. J Immunol 1997; 159: 3595–3602.

19.

Caramalho

Lopes-Carvalho

Ostler

Zelenay

Haury

Demengeot

. Regulatory T cells selectively express toll-like receptors and are activated by lipopolysaccharide. J Exp Med 2003; 197: 403–411.

20.

Danese

Mantovani

. Inflammatory bowel disease and intestinal cancer: a paradigm of the Yin-Yang interplay between inflammation and cancer. Oncogene 2010; 29: 3313–3323.

21.

Ivanov

McKenzie

Zhou

. The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17⁺ T helper cells. Cell 2006; 126: 1121–1133.

22.

Weaver

Harrington

Mangan

Gavrieli

Murphy

. Th17: an effector CD4 T cell lineage with regulatory T cell ties. Immunity 2006; 24: 677–688.

23.

Yamazaki

Matsuo

Muta

Yamamoto

Akira

Takeshige

. Gene-specific requirement of a nuclear protein, IκB-ζ, for promoter association of inflammatory transcription regulators. J Biol Chem 2008; 283: 32404–32411.

24.

Yan

Yang

Mody

. The adipokine lipocalin 2 is regulated by obesity and promotes insulin resistance. Diabetes 2007; 56: 2533–2540.

25.

Raices

Kannan

Bellamkonda-Athmaram

. A novel role for IκBζ in the regulation of IFNγ production. PLoS ONE 2009; 4: e6776–e6776.

26.

Okamoto

Iwai

Oh-Hora

. IκBζ regulates T(H)17 development by cooperating with ROR nuclear receptors. Nature 2010; 464: 1381–1385.

27.

Kawai

Adachi

Ogawa

Takeda

Akira

. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 1999; 11: 115–122.

28.

Libermann

Baltimore

. Activation of interleukin-6 gene expression through the NF-κB transcription factor. Mol Cell Biol 1990; 10: 2327–2334.

29.

Zhong

Voll

Ghosh

. Phosphorylation of NF-κB p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol Cell 1998; 1: 661–671.

30.

Anrather

Racchumi

Iadecola

. cis-acting, element-specific transcriptional activity of differentially phosphorylated nuclear factor-κB. J Biol Chem 2005; 280: 244–252.

31.

Cui

Tieu

Recinos

Tilton

Brasier

. RhoA mediates angiotensin II-induced phospho-Ser536 nuclear factor κB/RelA subunit exchange on the interleukin-6 promoter in VSMCs. Circ Res 2006; 99: 723–730.